This Breast Cancer Risk and Lifestyle (BCRL) study is a prospective large multicenter cohort study consisting of a consecutive case-series of breast cancer patients who were histopathologically diagnosed with primary malignancy. The study is designed to assess lifestyle-related factors in association with breast cancer risk prevention in northwestern Iran. The current study is a part of this cohort with ongoing recruitment, which began in May 2009. The case group consists of participants with newly diagnosed and histologically confirmed BrCa (n = 1007), who were admitted to the surgical wards of Noor-Nejat Hospital, Shams Hospital, Shahid Ghazi Educational-Oncology Hospital and several oncology clinics located in Tabriz, Iran from May 2009 to January 2018. These are referral hospitals for oncologic surgeries, with patients from different provinces in Northwestern Iran (East and West Azerbaijan, Ardabil, Hamadan, and Zanjan). The participants were native to these provinces or were long-term residents (i.e., had lived there for at least 10 years). Eligible cases were females mainly 27‒70 years of age (14 cases were 19‒27 years of age), with no previous history of neoplasm at another anatomic site, no history of cystic abnormalities and/or benign breast disease, and no history of adjuvant or neoadjuvant therapy (radiotherapy, chemotherapy, hormonal therapy, and trastuzumab). Patients at stage IV cancer was excluded, according to the TNM staging method (27). The controls were 1004 healthy women, mostly aged 27‒70 years of age (11 controls were 19‒27 years of age), admitted to the same hospitals during the same period, from hospital wards of orthopedics, ear-nose-throat, ophthalmology surgery, plastic surgery, and healthy women who were caregivers of these non-malignant patients. The controls were healthy women without any medical history of neoplasms, or disorders resulting in considerable metabolic changes. The controls were randomly selected and matched to cases on age at diagnosis (± 2 years) and the study region at a ratio of 1:1 (case: control).
The general inclusion criteria for both the case and control groups consisted of not following specific dietary patterns (vegetarian, DASH, homeopathy, and so on) within 12 months before BrCa diagnosis for the case group and interview for the control group; not currently pregnant, breastfeeding or postpartum at the time of enrollment; having no previous or recent occurrence of chronic inflammatory disorders (gastritis, colitis, multiple sclerosis, lupus erythematosus, and severe rheumatologic disorders); no former or recent occurrence of acute or chronic disease (such as severe liver or kidney failure, hyperthyroidism, and polycystic ovary syndrome); and not being a long-term consumer of aspirin, glucocorticoids, methotrexate, anticonvulsants, sulfasalazine, and contraceptive drugs. The participant with an oophorectomy history reflected the early menopausal status and was excluded. Very active or sedentary subjects and those with morbid obesity (BMI > 45 kg/m2) were excluded from the present study.
General data
A face-to-face interview for each participant was conducted before the surgery by an expert interviewer using a structured questionnaire. The general questionnaire consisted of items regarding sociodemographic characteristics, reproductive variables, medical history, family history of BrCa, and records of dietary supplements and medicines.
Histopathological data
Pathological data for BrCa patients, including histological subtypes (invasive-ductal or invasive-lobular carcinoma), histological grade (I, II, III), tumor size (cm) and immunohistochemistry (IHC) data, including estrogen receptor (ER), progesterone receptor (PR) and human epidermal growth factor receptor 2 (HER2-neu) were collected by reviewing the medical records of patients’ pathological examinations. The pathologist fixed the tumor specimens in 10% buffered formalin and prepared formalin-fixed paraffin-embedded tumor blocks. The IHC staining was conducted through the application of antibodies on tumor sections, primarily deparaffinized and incubated at 8ºC overnight. The percentage of stained cells by the antibodies was examined using a binocular microscope (Zeiss KF2 binocular, Germany). HER2 positivity was determined when the membrane/membrane plus cytoplasm stained by antibody (A0485, 1/200; Dako Denmark A/S) accounted for ≥ 10% of tumor cells (28). The Clone ID5 (Dako Denmark A/S) antibody (Glostrup, Denmark) was applied to detect the frequency of cells with ER positivity. Similarly, the IHC staining using Clone PgR636 (Dako Denmark A/S) antibody (Glostrup, Denmark) was applied for PR staining. The protein expression status of ER and PR > 5% were taken into account to identify the positivity of HR (29). The BrCa phenotypes were classified into four molecular subtypes based on IHC data, according to Parise et al. (30) as follows: luminal A (ER + and/or PR+, HER2-), luminal B (ER + and/or PR+, HER2+), triple-negative (ER-, PR-, HER2-), and HER2-enriched (ER-, PR-, HER2+).
Nutritional data
Dietary data were collected using a validated semi-quantitative 136-item food frequency questionnaire (FFQ) (31–34). Portion sizes and frequency of consumption were requested for each food item on a daily, weekly, monthly, or yearly basis. The portion size was defined based on common household utensils provided for each food item. Food portion sizes were converted to grams using standard reference values (35). The timing of FFQ questioning covered up to a year before diagnosis for cases or the control’s hospital admission. The intake levels of total energy and nutrients were obtained using Nutritionist IV software (version 3.5.2; 1994, N-Squared Computing, San Bruno, CA, USA) (31). The flavonoid contents of each food and beverage for five major classes of flavonoids (flavanols, flavones, flavonols, flavanones, and anthocyanidins) were calculated using a database developed by the US Department of Agriculture (36).
Dietary inflammatory index (DII®)
The DII is a tool to assess the inflammatory effects of the diet, which was generated by articles assessing the effect of specific food parameters on inflammatory biomarkers (12). Briefly, DII development was carried out by reviewing and scoring the eligible articles of different type of studies (1943 articles), which investigated diet (foods and dietary ingredients) in association with inflammatory biomarkers such as Interleukins 1β, 4, 6, and 10 (IL-1β, IL-4, IL-6, and IL-10), tumor necrosis factor-α (TNF-α), and C-reactive protein (CRP). The specific inflammatory scores were computed for all of the constituents of DII, i.e., up to 45 dietary parameters, which included macronutrients, micronutrients, and bioactive components, such as spices, tea, and flavonoids. The estimation of the global mean intake and global standard deviation of each food parameter was calculated using 11 dietary datasets from different countries around the world. The dietary data of each participant was standardized to the global database and exposure relative to the global mean defined as a z-score. The z-score was calculated by subtracting the global mean from the mean of reported dietary intake. For this study, we used the energy-adjusted DII (E-DII™), which describes exposure per 1000 calories for each food parameter. To compute E-DII scores, we used an energy-adjusted global comparative database. To decrease the right skewing, the z-score value of each food parameter was converted to a proportion and this was centered by doubling and subtracting 1. Finally, to obtain the overall E-DII score for each subject, all of the individual E-DII scores for each food parameter were added together. In the present study, data were available for 39 of the 45 food parameters assigned as a component of DII, including energy, carbohydrate, protein, fiber, total fat, monounsaturated fat, polyunsaturated fat, saturated fat, n-3 fatty acids, n-6 fatty acids, cholesterol, vitamin A, vitamin D, vitamin E, thiamin, riboflavin, niacin, vitamin B6, vitamin B12, vitamin C, folic acid, b-carotene, iron, zinc, magnesium, selenium, caffeine, green/black tea, garlic, onion, saffron, turmeric, pepper, ginger, flavan-3-ol, flavones, flavonols, flavanones, and anthocyanidins. Food items consisting of isoflavones, thyme, rosemary, trans-fat, eugenol, and caffeine were not included in the DII calculation. The inflammatory effect of energy was not considered in E-DII calculation, as the score itself accounted for energy. Moreover, we computed the E-DII both with and without considering the inflammatory effects of dietary supplements. In the present study, the dietary supplements included in the calculation of DII (i.e., DII-s) and E-DII (i.e., E-DII-s) scores consisted of vitamins A, B1 (thiamin), B2 (riboflavin), B3 (niacin), B6 (pyridoxine), B12 (cobalamin), C (ascorbic acid), B9 (folic acid), D (cholecalciferol), E (α-tocopherol), and K, and iron, zinc, selenium, and magnesium.